The present invention concerns methods and compositions for inhibiting neoplastic cell growth. In particular, the present invention concerns antitumor compositions and methods for the treatment of tumors.
Apoptosis is a form of programmed cell death which occurs through the activation of cell-intrinsic suicide machinery. The biochemical machinery responsible for apoptosis is expressed in most, if not all, cells. Apoptosis is primarily a physiologic process necessary to remove individual cells that are no longer needed or that function abnormally. Apoptosis is a regulated event dependent upon active metabolism and protein synthesis by the dying cell.
The morphological and biochemical characteristics of cells dying by apoptosis differ markedly from those of cells dying by necrosis. During apoptosis, cells decrease in size and round up. The nuclear chromatin undergoes condensation and fragmentation. Cell death is preceded by DNA fragmentation. The DNA of apoptotic cells is nonrandomly degraded by endogenous calcium and magnesium-dependent endonuclease(s) inhibited by zinc ions. This enzyme(s) gives fragments of approx. 200 base pairs (bp) or multiples of 200 bp by cutting the linker DNA running between nucleosomes. Thus DNA appears to be one of the most important targets of the process that leads to cell suicide. The apoptotic cell then breaks apart into many plasma membrane-bound vesicles called xe2x80x9capoptotic bodies,xe2x80x9d which contain fragments of condensed chromatin and morphologically intact organelles such as mitochondria. Apoptotic cells and bodies are rapidly phagocytosed, thereby protecting surrounding tissues from injury. The rapid and efficient clearance of apoptotic cells makes apoptosis extremely difficult to detect in tissue sections.
In contrast, necrosis is associated with rapid metabolic collapse that leads to cell swelling, early loss of plasma membrane integrity, and ultimate cell rupture. Cytosolic contents leach from the necrotic cell causing injury and inflammation to surrounding tissue.
In contrast to the cell death caused by cell injury, apoptosis is an active process of gene-directed, cellular self-destruction and that it serves a biologically meaningful function. (Kerr, J. F. R and J. Searle. J. Pathol. 107:41, 1971). Apoptosis plays a key role in the human body from the early stages of embryonic development through to the inevitable decline associated with old age. (Wyllie, A. H. Int. Rev. Cytol. 68:251, 1980). The normal function of the immune, gastrointestinal and hematopoietic system relies on the normal function of apoptosis. When the normal function of apoptosis goes awry, the cause or the result can be one of a number of diseases, including: cancer, viral infections, auto-immune disease/allergies, neurodegeneration or cardiovascular diseases. Because of the versatility of apoptosis involved in human diseases, apoptosis is becoming a prominent buzzword in the pharmaceutical research field.
The idea of modulating apoptosis as a means of treating and/or preventing cancer is a relatively new idea (Cope, F. O and Wille, J. Apoptosis: The Molecular Basis of Cell Death. Cold Spring Harbor Laboratory Press, p. 61, 1991). Apoptosis modulation is a potential mechanism for controlling the growth of tumor cells without the side effects of many current cancer treatment regimes. In addition to cancer, recent studies show that multiple cytotoxic stimuli well known to cause necrosis can lead to apoptosis instead when cells are exposed to the same noxious agents at lower concentrations.
Malignant tumors (cancers) are the second leading cause of death in the United States, after heart disease (Boring et al., CA Cancel J. Clin., 43:7 (1993)).
Cancer is characterized by the increase in the number of abnormal, or neoplastic, cells derived from a normal tissue which proliferate to form a tumor mass, the invasion of adjacent tissues by these neoplastic tumor cells, and the generation of malignant cells which eventually spread via the blood or lymphatic system to regional lymph nodes and to distant sites (metastasis). In a cancerous state a cell proliferates under conditions in which the normal cells would not grow. Cancer manifests itself in a wide variety of forms, characterized by different degrees of invasiveness and aggressiveness.
Despite recent advances in cancer therapy, there is a great need for new therapeutic agents capable of inhibiting neoplastic cell growth. Accordingly, an objective of the present invention is methods and compositions capable of inhibiting the growth of neoplastic cells, such as cancer cells, by inducing apoptosis and necrosis.
The present invention is relates to embodiments including, but not limited to, GSSP-2 polypeptides, polynucleotides encoding GSSP-2 polypeptides, vectors comprising GSSP-2 polynucleotides, and cells comprising GSSP-2 polynucleotides, as well as to pharmaceutically and physiologically acceptable compositions comprising GSSP-2 polypeptides and methods of contacting neoplastic cells with GSSP-2 polypeptides to suppress tumor growth.
In particular, the present invention relates to methods and compositions for inhibiting neoplastic cell growth, killing neoplastic cells and treating cancer. More particularly, the invention concerns methods and compositions to inhibit cellular proliferation of neoplastic cells, induce cytotoxicity in neoplastic cells and kill neoplastic cells. These properties thus make GSSP-2 useful in the treatment neoplastic disease, including cancers, such as breast, prostate, colon, ovarian, renal, liver and CNS cancers, leukemia, lymphoma, sarcoma, melanoma, etc., preferably liver cancers, in mammalian patients, preferably humans.
A first embodiment of the invention is a recombinant, purified or isolated polynucleotide comprising, or consisting of a mammalian genomic sequence, gene, or fragments thereof. In one aspect the sequence is derived from a human, mouse or other mammal. In a preferred aspect, the genomic sequence includes isolated, purified, or recombinant polynucleotides comprising a contiguous span of at least 12, 15, 18, 20, 22, 25, 30, 35, 40, 50, 60, 70, 80, 90, 100, 150, 200, 500, 1000,2000, 5000, 10000 or 50000 nucleotides of SEQ ID NO: 1, or the complements thereof, wherein said contiguous span comprises at least 1, 2, 3, 5, 6, 7 or 8 of the following nucleotide positions of SEQ ID NO: 1: 739-1739; 10946-12958; 13470-13526; 13641-13752; 14271-17969; 41718-42718; 44942-45942; and 76558-77558. Further preferred nucleic acids of the invention include isolated, purified, or recombinant polynucleotides comprising a contiguous span of at least 12, 15, 18, 20, 25, 30, 35, 40, 50, 60, 70, 80, 90, 100, 150, 200, 500, or 1000 nucleotides of SEQ ID NO: 1, or the complements thereof, wherein said contiguous span contains one or more of the nucleotides at positions 1239, 12347, 15241, 42218, 45442, or 77058. Optionally, the polynucleotide consists of, consists essentially of, or comprises a contiguous span of nucleotides of a human genomic sequence, preferably a sequence selected from SEQ ID NO: 1, wherein said contiguous span is at least 6, 8, 10, 12, 15, 20, 25, 30, 50, 100, 200, 500 or 1000 nucleotides in length and contains one or more of the nucleotides at positions 13269 or 13475.
Another embodiment of the invention is a recombinant, purified or isolated polynucleotide comprising, or consisting of a mammalian genomic sequence, gene, or fragments thereof. In one aspect the sequence is derived from a human, mouse or other mammal. In a preferred aspect, the genomic sequence is selected from the human genomic sequence of SEQ ID NO: 4. Optionally, the polynucleotide consists of, consists essentially of, or comprises a contiguous span of nucleotides of a human genomic sequence, preferably a sequence selected from SEQ ID NO: 4, wherein said contiguous span is at least 6, 8, 10, 12, 15, 20, 25, 30, 50, 100, 200, 500, 1000, 2000, 3000, 4000 or 5000 nucleotides in length and contains one or more of the nucleotides at positions 1241 or 1447. Optionally, the polynucleotide consists of, consists essentially of, or comprises a contiguous span of nucleotides of a human genomic sequence, preferably SEQ ID NO: 4, wherein said contiguous span comprises at least 6, 8, 10, 12, 15, 20, 25, 30, 50, 100, 200, 500 or 1000 nucleotides of the following nucleotide positions of SEQ ID NO: 4: 1-1498, 1613-1724, 2243-3940, and 3941-5381.
Another embodiment of the present invention is a recombinant, purified or isolated polynucleotide comprising, or consisting of a mammalian cDNA sequence, or fragments thereof. In one aspect the sequence is derived from a human, mouse or other mammal. In a preferred aspect, the cDNA sequence is selected from the human cDNA sequence of SEQ ID NO: 2 or the complement thereto. Optionally, said polynucleotide consists of, consists essentially of, or comprises a contiguous span of nucleotides of a mammalian cDNA sequence, preferably SEQ ID NO: 2. Preferred fragments of said cDNA include the fragments delineated by the exons of SEQ ID NO:4 (1-1498, 1613-1724, 2243-3940 and 3941-5381).
A further embodiment of the present invention is a recombinant, purified or isolated polynucleotide, or the complement thereof, encoding a mammalian GSSP-2 protein, fragment thereof or other polypeptide of the present invention. In one aspect the GSSP-2 protein sequence is from a human, mouse or other mammal. In a preferred aspect, the GSSP-2 protein sequence is selected from the human GSSP-2 protein sequence of SEQ ID NO: 3. Optionally, said fragment of GSSP-2 polynucleotide consists of, consists essentially of, or comprises a nucleic acid sequence encoding a contiguous stretch of at least 8, 10, 12, 15, 20, 25, 30, 50, 100, 200, 300 or 350 amino acids from SEQ ID NO: 3, as well as any other human, mouse or mammalian GSSP-2 polypeptide of the present invention. The invention further includes polypeptides and isolated nucleic acid molecules encoding such polypeptides, including mRNAs, DNAs, cDNAs, genomic DNA as well as biologically active and diagnostically or therapeutically useful fragments, analogs and derivatives thereof.
A further embodiment of the invention is a purified or isolated mammalian GSSP-2 gene or cDNA sequence, or polynucleotide encoding a mammalian GSSP-2 polypeptide or fragment thereof.
An embodiment of the invention is the polynucleotide primers and probes disclosed herein.
An embodiment of the present invention is a recombinant, purified or isolated polypeptide comprising or consisting of a mammalian GSSP-2 protein, or a fragment thereof. In one aspect the GSSP-2 protein sequence is from a human, mouse or other mammal. In a preferred aspect, the GSSP-2 protein sequence is selected from the human GSSP-2 protein sequence of SEQ ID NO: 3. Optionally, said fragment of GSSP-2 polypeptide consists of, consists essentially of, or comprises a contiguous stretch of at least 8, 10, 12, 15, 20, 25, 30, 50, 100, 200, 300 or 350 amino acids from SEQ ID NO: 3, as well as any other human, mouse or mammalian GSSP-2 polypeptide. The invention further includes polypeptides and isolated nucleic acid molecules encoding such polypeptides, including mRNAs, DNAs, cDNAs, genomic DNA as well as biologically active and diagnostically or therapeutically useful fragments, analogs and derivatives thereof. The invention also includes a chimeric molecule comprising a polypeptide fused to a heterologous amino acid sequence.
Another embodiment of the invention encompasses any polynucleotide or polypeptide of the invention attached to a solid support. In addition, the polynucleotides or polypeptides of the invention which are attached to a solid support encompass polynucleotides or polypeptides with any further limitation described in this disclosure. Optionally, said polynucleotides or polypeptides are specified as attached individually or in groups of at least 2, 5, 8, 10, 12, 15, 20, or 25 distinct polynucleotides of the inventions to a single solid support. Optionally, when multiple polynucleotides or polypeptides are attached to a solid support they are attached at random locations, or in an ordered array. Optionally, said ordered array is addressable.
Another embodiment of the present invention is an antibody composition capable of specifically binding to a polypeptide of the invention. Optionally, said antibody is polyclonal or monoclonal. Optionally, said polypeptide is an epitope-containing fragment of at least 8, 10, 12, 15, 20, 25, or 30 amino acids of a human, mouse, or mammalian GSSP-2 protein, preferably a sequence selected from SEQ ID NO: 3.
A further embodiment of the present invention is a vector comprising any polynucleotide of the invention. Optionally, said vector is a cloning vector, an expression vector, gene therapy vector, amplification vector, gene targeting vector, or knock-out vector.
A further embodiment of the present invention is a host cell recombinant for any vector or polynucleotide of the invention.
A further embodiment of the present invention is a mammalian host cell comprising a GSSP-2 regulatory region (e.g., 5xe2x80x2 promoter) or exonic or intronic or any combination thereof altered or disrupted by homologous recombination with a knock out or knock in vector.
A further embodiment of the present invention is a nonhuman host mammal or animal comprising a polynucleotide of the invention.
In another related aspect, the invention features a cell that is recombinant for a polynucleotide encoding a GSSP-2 polypeptide of the invention. In a preferred embodiment of this aspect, the polynucleotide is expressed in the cell. In various preferred embodiments, the cell is present in a patient having a disease that is caused by excessive cell growth or insufficient cell death and the cell is selected from the group that includes bladder carcinoma, hepatocarcinoma, hepatoblastoma, rhabdomyosarcoma, ovarian carcinoma, cervical carcinoma, lung carcinoma, breast carcinoma, squamous cell carcinoma in head and neck, esophageal carcinoma, thyroid carcinoma, astrocytoma, ganglioblastoma, neuroblastoma, lymphoma, myeloma, sarcoma and neuroepithelioma.
An embodiment of the present invention is a transgenic animal generated from a cell genetically engineered to lack nucleic acid molecule encoding a GSSP-2 polypeptide, where the transgenic animal lacks expression of the GSSP-2 polypeptide.
In a related aspect, the invention features a transgenic animal generated from a cell that contains a substantially pure nucleic acid molecule that replaces DNA encoding a GSSP-2 polypeptide, where the nucleic acid molecule is expressed in the transgenic animal.
An embodiment of the present invention includes the nucleic acid and amino acid sequences of mutant or low frequency GSSP-2 alleles derived from neoplastic patients, tissues or cell lines. The present invention also encompasses methods which utilize detection of these mutant GSSP-2 sequences in an individual or tissue sample to diagnosis a neoplastic disease, assess the risk of developing a neoplastic disease or assess the likely severity of said disorder. An embodiment of the present invention is a method of obtaining an allele of the GSSP-2 gene which is associated with a detectable phenotype comprising obtaining a nucleic acid sample from an individual expressing the detectable phenotype, contacting the nucleic acid sample with an agent capable of specifically detecting a nucleic acid molecule encoding the GSSP-2 protein, and isolating the nucleic acid molecule encoding the GSSP-2 protein. In one aspect of this method, the contacting step comprises contacting the nucleic acid sample with at least one nucleic acid probe capable of specifically hybridizing to said nucleic acid molecule encoding the GSSP-2 protein. In another aspect of this embodiment, the contacting step comprises contacting the nucleic acid sample with an antibody capable of specifically binding to the GSSP-2 protein. In another aspect of this embodiment, the step of obtaining a nucleic acid sample from an individual expressing a detectable phenotype comprises obtaining a nucleic acid sample from an individual suffering from a neoplastic disease.
Another embodiment of the present invention is a method of obtaining an allele of the GSSP-2 gene which is associated with a detectable phenotype comprising obtaining a nucleic acid sample from an individual expressing the detectable phenotype, contacting the nucleic acid sample with an agent capable of specifically detecting a sequence within the 11 q23 region of the human genome, identifying a nucleic acid molecule encoding the GSSP-2 protein in the nucleic acid sample, and isolating the nucleic acid molecule encoding the GSSP-2 protein. In one aspect of this embodiment, the nucleic acid sample is obtained from an individual suffering from a neoplastic disease (e.g., cancer).
A further embodiment of the invention encompasses methods of genotyping a biological sample comprising determining the identity of an allele at an GSSP-2-related biallelic marker. In addition, the genotyping methods of the invention encompass methods with any further limitation described in this disclosure, or those following: Optionally, said GSSP-2-related biallelic marker is a GSSP-2-related biallelic marker positioned in SEQ ID NOs: 1, 2 or 4; one or more GSSP-2-related biallelic marker selected from the group consisting of 20-828-311, 17-42-319, 17-41-250, 20-841-149, 20-842-115, and 20-853-415; or more preferably a GSSP-2-related biallelic mar selected from the group consisting of 1742-319 and 17-41-250. Optionally, said method further comprises determining the identity of a second allele at said biallelic marker, wherein said first allele and second allele are not base paired (by Watson and Crick base pairing) to one another. Optionally, said biological sample is derived from a single individual or subject. Optionally, said method is performed in vitro. Optionally, said biallelic marker is determined for both copies of said biallelic marker present in said individual""s genome. Optionally, said biological sample is derived from multiple subjects or individuals. Optionally, said method further comprises amplifying a portion of said sequence comprising the biallelic marker prior to said determining step. Optionally, wherein said amplifying is performed by PCR, LCR, or replication of a recombinant vector comprising an origin of replication and said portion in a host cell. Optionally, wherein said determining is performed by a hybridization assay, sequencing assay, microsequencing assay, or allele-specific amplification assay.
An additional embodiment of the invention comprises methods of estimating the frequency of an allele in a population comprising determining the proportional representation of an allele at a GSSP-2-related biallelic marker in said population. In addition, the methods of estimating the frequency of an allele in a population of the invention encompass methods with any further limitation described in this disclosure, or those following: Optionally, said GSSP-2-related biallelic marker is a GSSP-2-related biallelic marker positioned in SEQ ID NOs: 1, 2 or 4; one or more GSSP-2-related biallelic marker selected from the group consisting of 20-828-311, 17-42-319, 17-41-250, 20-841-149, 20-842-115, and 20-853-415; or more preferably a GSSP-2-related biallelic marker selected from the group consisting of 17-42-319 and 17-41-250. Optionally, determining the proportional representation of an allele at a GSSP-2-related biallelic marker is accomplished by determining the identity of the alleles for both copies of said biallelic marker present in the genome of each individual in said population and calculating the proportional representation of said allele at said GSSP-2-related biallelic marker for the population. Optionally, determining the proportional representation is accomplished by performing a genotyping method of the invention on a pooled biological sample derived from a representative number of individuals, or each individual, in said population, and calculating the proportional amount of said nucleotide compared with the total.
A further embodiment of the invention comprises methods of detecting an association between a genotype and a phenotype, comprising the steps of a) genotyping at least one GSSP-2-related biallelic marker in a trait positive population according to a genotyping method of the invention; b) genotyping said GSSP-2-related biallelic marker in a control population according to a genotyping method of the invention; and c) determining whether a statistically significant association exists between said genotype and said phenotype. In addition, the methods of detecting an association between a genotype and a phenotype of the invention encompass methods with any further limitation described in this disclosure, or those following: SEQ ID NOs: 1, 2 or 4; one or more GSSP-2-related biallelic marker selected from the group consisting of 20-828-311, 17-42-319, 17-41-250, 20-841-149, 20-842-115, and 20-853-415; or more preferably a GSSP-2-related biallelic marker selected from the group consisting of 17-42-319 and 17-41-250. Optionally, said control population is a trait negative population, or a random population. Optionally, each of said genotyping steps a) and b) is performed on a single pooled biological sample derived from each of said populations. Optionally, each of said genotyping of steps a) and b) is performed separately on biological samples derived from each individual in said population or a subsample thereof. Optionally, said phenotype is a neoplastic disease; a response to an agent acting on lipid metabolism and/or liver related disorders; or a side effect to an agent acting on lipid metabolism. Optionally, said method comprises the additional steps of determining the phenotype in said trait positive and said control populations prior to step c).
An additional embodiment of the present invention encompasses methods of estimating the frequency of a haplotype for a set of biallelic markers in a population, comprising the steps of: a) genotyping at least one GSSP-2-related biallelic marker for both copies of said set of biallelic marker present in the genome of each individual in said population or a subsample thereof, according to a genotyping method of the invention; b) genotyping a second biallelic marker by determining the identity of the allele at said second biallelic marker for both copies of said second biallelic marker present in the genome of each individual in said population or said subsample, according to a genotyping method of the invention; and c) applying a haplotype determination method to the identities of the nucleotides determined in steps a) and b) to obtain an estimate of said frequency. In addition, the methods of estimating the frequency of a haplotype of the invention encompass methods with any further limitation described in this disclosure, or those following: Optionally, said GSSP-2-related biallelic marker is a GSSP-2-related biallelic marker positioned in SEQ ID NOs: 1, 2 or 4; one or more GSSP-2-related biallelic marker selected from the group consisting of 20-828-311, 17-42-319, 17-41-250, 20-841-149, 20-842-115, and 20-853-415; or more preferably a GSSP-2-related biallel marker selected from the group consisting of 17-42-319 and 1741-250. Optionally, said haplotype determination method is an expectation-maximization algorithm.
An additional embodiment of the present invention encompasses methods of detecting an association between a haplotype and a phenotype, comprising the steps of: a) estimating the frequency of at least one haplotype in a trait positive population, according to a method of the invention for estimating the frequency of a haplotype; b) estimating the frequency of said haplotype in a control population, according to a method of the invention for estimating the frequency of a haplotype; and c) determining whether a statistically significant association exists between said haplotype and said phenotype. In addition, the methods of detecting an association between a haplotype and a phenotype of the invention encompass methods with any further limitation described in this disclosure, or those following: Optionally, said GSSP-2-related biallelic is a GSSP-2-related biallelic marker positioned in SEQ ID NOs: 1, 2 or 4; one or more GSSP-2-related biallelic marker selected from the group consisting of 20-828-311, 17-42-319, 17-41-250, 20-841-149, 20-842-115, and 20-853-415; or more preferably a GSSP-2-related biallelic marker selected from the group consisting of 17-42-319 and 17-41-250. Optionally, said haplotype exhibits a p-value of less than 1xc3x9710xe2x88x923 in an association with a trait positive population with a disorder, preferably a neoplastic disease. Optionally, said control population is a trait negative population, or a random population. Optionally, said phenotype is a neoplastic disease; a response to an agent acting on a neoplastic disease; or a side effect to an agent acting on a neoplastic disease. Optionally, said method comprises the additional steps of determining the phenotype in said trait positive and said control populations prior to step c).
Another embodiment of the present invention comprises a method of identifying molecules which specifically bind to a GSSP-2 protein, preferably the protein of SEQ ID NO: 3 or a portion thereof: comprising the steps of introducing a nucleic a nucleic acid molecule encoding the protein of SEQ ID NO: 3 or a portion thereof into a cell such that the protein of SEQ ID NO: 3 or a portion thereof contacts proteins expressed in the cell and identifying those proteins expressed in the cell which specifically interact with the protein of SEQ ID NO: 3 or a portion thereof.
Another embodiment of the present invention is a method of identifying molecules which specifically bind to the protein of SEQ ID NO: 3 or a portion thereof. One step of the method comprises linking a first nucleic acid molecule encoding the protein of SEQ ID NO: 3 or a portion thereof to a first indicator nucleic acid molecule encoding a first indicator polypeptide to generate a first chimeric nucleic acid molecule encoding a first fusion protein. The first fusion protein comprises the protein of SEQ ID NO: 3 or a portion thereof and the first indicator polypeptide. Another step of the method comprises linking a second nucleic acid molecule encoding a test polypeptide to a second indicator nucleic acid molecule encoding a second indicator polypeptide to generate a second chimeric nucleic acid molecule encoding a second fusion protein. The second fusion protein comprises the test polypeptide and the second indicator polypeptide. Association between the first indicator protein and the second indicator protein produces a detectable result. Another step of the method comprises introducing the first chimeric nucleic acid molecule and the second chimeric nucleic acid molecule into a cell. Another step comprises detecting the detectable result.
An embodiment of the present invention is a method of identifying a compound that modulates apoptosis and/or necrosis. The method includes: (a) providing a cell that has a GSSP-2 gene; (b) contacting the cell with a candidate compound; and (c) monitoring expression of the GSSP-2 gene, where an alteration in the level of expression of the GSSP-2 gene indicates the presence of a compound which modulates apoptosis and/or necrosis. In one preferred embodiment of this aspect, the alteration that is an increase of GSSP-2 mRNA or protein indicates the compound is increasing apoptosis or necrosis, and the alteration that is a decrease indicates the compound is decreasing apoptosis and/or necrosis. In various embodiments of this aspect, the cell is transformed and the cell is not able to induce apoptosis and/or necrosis.
In a related aspect, the invention features another method of identifying a compound that is able to modulate apoptosis and/or necrosis that includes: (a) providing a cell including a reporter gene operably linked to a promoter from a GSSP-2 gene; (b) contacting the cell with a candidate compound; and (c) measuring expression of the reporter gene, where a change in the expression in response to the candidate compound identifies a compound that is able to modulate apoptosis and/or necrosis. In one preferred embodiment of this aspect, the alteration that is an increase in reporter gene activity indicates the compound is increasing apoptosis and/or necrosis, and the alteration that is a decrease indicates the compound is decreasing apoptosis and/or necrosis.
An embodiment of the present invention is a method of identifying a compound that is able to inhibit GSSP-2-mediated apoptosis and/or necrosis that includes: (a) providing a cell expressing or contacted with an apoptosis and/or necrosis-inducing amount of GSSP-2; (b) contacting the cell with a candidate compound; and (c) measuring the level of apoptosis and/or necrosis in the cell, where a decrease in the level of apoptosis and/or necrosis relative to a level of apoptosis and/or necrosis in a cell not contacted with the candidate compound indicates a compound that is able to inhibit GSSP-2-mediated apoptosis and/or necrosis. In various embodiments of this aspect, the cell is transformed and the cell is not able to induce apoptosis and/or necrosis.
An embodiment of the present invention is a method of identifying a compound that is able to induce GSSP-2-mediated apoptosis and/or necrosis that includes: (a) providing a cell expressing or contacting with an apoptosis and/or necrosis-inducing amount of GSSP-2; (b) contacting the cell with a candidate compound; and (c) measuring level of apoptosis and/or necrosis in the cell, where an increase in the level relative to a level in a cell not contacted with the candidate compound indicates a compound that able to induce GSSP-2-mediated apoptosis and/or necrosis. In various embodiments of this aspect, the cell is transformed and the cell is not able to induce apoptosis and/or necrosis.
A further embodiment of the present invention is a method of inducing apoptosis and/or necrosis in a cell by contacting the cell with an apoptosis and/or necrosis inducing amount of GSSP-2 polypeptide or fragment thereof.
In related aspects, the invention includes methods of inducing apoptosis and/or necrosis by either providing a transgene encoding a GSSP-2 polypeptide or fragment thereof to a cell of an animal such that the transgene is positioned for expression in the cell; or by administering to the cell a compound which increases GSSP-2 biological activity in a cell.
An embodiment of the invention is a method of inhibiting the cellular proliferation of a neoplastic cell comprising: (a) contacting said cell with an effective amount of a polypeptide of SEQ ID NO: 3 or a polypeptide encoded by the human cDNA of clone 117-005-2-0-E10-FLC, or an apoptosis or cytotoxicity inducing polypeptide fragment of SEQ ID NO: 3 or clone 117-005-2-0-E10-FLC. In another aspect of the invention, said neoplastic cell is selected from the group consisting of a hepatocellular carcinoma cell and a lymphoma cell. In another aspect of the invention, said neoplastic cell is a transformed cell. In yet another aspect of the invention, said neoplastic cells are from a malignant tumor or benign tumor.
Another embodiment of the invention is a method of preferentially inhibiting the cellular proliferation of a neoplastic cell compared to a normal cell comprising: (a) contacting said cell with an effective amount of a polypeptide of the present invention or a polypeptide encoded by the human cDNA of clone 117-005-2-0-E10-FLC, or an apoptosis or cytotoxicity inducing polypeptide fragment of SEQ ID NO: 3 or clone 117-005-2-0-E10-FLC. In a preferred aspect of the invention, said neoplastic cell is selected from the group consisting of hepatocellular carcinoma cell and a lymphoma cell. In another aspect of the invention, said neoplastic cell is a transformed cell. In yet another aspect of the invention, said neoplastic cell is a cell of a malignant or benign tumor.
Another embodiment of the invention is a method of inducing cytotoxicity in a neoplastic cell comprising: (a) contacting said cell with an effective amount of a polypeptide of SEQ ID NO: or a polypeptide encoded by the human cDNA of clone 1 17-005-2-0-E10-FLC, or a cytotoxicity-inducing polypeptide fragment of SEQ ID NO: 3 or clone 1 17-005-2-0-E10-FLC. In one aspect of the invention, inducing cytotoxicity refers to inducing apoptosis. In another aspect, inducing cytotoxicity refers to inducing necrosis. In another aspect of the invention, said neoplastic cell is selected from the group consisting of hepatocellular carcinoma cell and a lymphoma cell. In another aspect of the invention, said neoplastic cell is a transformed cell. In yet another aspect of the invention, said neoplastic cell is a cell of a malignant or benign tumor.
Another embodiment of the invention is a method of preferentially inducing cytotoxicity in a neoplastic cell compared to a normal cell comprising: (a) contacting said cell with an effective amount of a polypeptide of SEQ ID NO: 3 or a polypeptide encoded by the human cDNA of clone 117-005-2-0-E10-FLC, or an cytotoxicity inducing polypeptide fragment of SEQ ID NO: 3 or clone 11 7-005-2-0-E10-FLC. In one aspect of the invention, inducing cytotoxicity refers to inducing apoptosis. In another aspect, inducing cytotoxicity refers to inducing necrosis. In another aspect of the invention, said neoplastic cell is selected from the group consisting of hepatocellular carcinoma cell and a lymphoma cell. In another aspect of the invention, said neoplastic cell is a transformed cell. In yet another aspect of the invention, said neoplastic cell is a cell of a malignant or benign tumor.
In preferred embodiment, the GSSP-2 is from a mammal (e.g., a human or rodent); the cell is in a mammal (e.g., a human or rodent); the cell is in a mammal diagnosed or suspected as having a condition involving neoplastic cell growth, (e.g., a cancer such as prostate cancer, skin cancer, pancreatic carcinoma, colon cancer, melanoma, ovarian cancer, liver cancer, small cell lung carcinoma, non-small cell lung carcinoma, cervical cancer, breast cancer, bladder cancer, brain cancer, neuroblastoma/glioblastoma, leukemia, head and neck cancer, kidney cancer, lymphoma, myeloma and ovarian cancer).
Another embodiment of the invention is a method of suppressing tumor growth comprising: (a) contacting said tumor with an effective amount of a polypeptide of SEQ ID NO: 3 or a polypeptide encoded by the human cDNA of clone 117-005-2-0-E10-FLC, or an apoptosis and/or necrosis inducing polypeptide fragment of SEQ ID NO: 3 or clone 117-005-2-0-E10-FLC. The method of suppressing tumor growth comprises the effects selected from the group consisting of: (a) inhibiting cell growth or proliferation in said tumor; (b) killing cells in said tumor; (c) inducing apoptosis in said tumor; (d) inducing necrosis in said tumor; (e) preventing or inhibiting tumor cell invasion; and (f) preventing or inhibiting tumor cell metastasis. In another aspect of the invention, said tumor is selected from the group consisting of bladder carcinoma, hepatocarcinoma, hepatoblastoma, rhabdomyosarcoma, ovarian carcinoma, cervical carcinoma, lung carcinoma, breast carcinoma, squamous cell carcinoma in head and neck, esophageal carcinoma, thyroid carcinoma, astrocytoma, ganglioblastoma, neuroblastoma, lymphoma, myeloma, sarcoma and neuroepithelioma. In yet another aspect of the invention, said tumor is malignant or benign.
An embodiment of the present invention is a method of treating a patient having a neoplastic disease (e.g., cancer) characterized by proliferation of neoplastic cells which comprises administering to the patient an amount of a polypeptide of the invention, effective to: (a) selectively induce apoptosis and/or necrosis in such neoplastic cells and thereby inhibit their proliferation; (b) inhibit cell growth and proliferation of the neoplastic cells; (c) inhibit invasion of the neoplastic cells; (d) inhibit metastasis of the neoplastic cells; (e) kill neoplastic cells; (g) preferentially inhibit cell growth and proliferation of the neoplastic cells; and (h) preferentially kill neoplastic cells.
Another embodiment of the present invention features a method of treating a neoplastic disease in an individual comprising administering to an individual in need of such treatment an GSSP-2 polypeptide of the invention in a pharmaceutically or physiologically acceptable composition such as a composition comprising a carrier. Alternatively, antagonists or agonists of GSSP-2 activity can be provided, or compounds that enhance or inhibit the expression of GSSP-2.
The present invention further relates to methods of preferentially killing neoplastic cells and treating diseases/disorders such as cancer, (e.g., prostate cancer, skin cancer, pancreatic carcinoma, colon cancer, melanoma, ovarian cancer, liver cancer, small cell lung carcinoma, non-small cell lung carcinoma, cervical cancer, breast cancer, bladder cancer, brain cancer, neuroblastoma/glioblastoma, leukemia, head and neck cancer, kidney cancer, lymphoma, myeloma and ovarian cancer).
The present invention also relates to pharmaceutical or physiologically acceptable compositions comprising, an active agent, the polypeptides, polynucleotide or antibodies of the present invention. A preferred composition further comprises a carrier.
The present invention relates to an article of manufacture comprising: (a) a container; and (b) a composition comprising an active agent contained within the container; wherein said active agent in the composition is a GSSP-2 polypeptide, or an agonist thereof. A preferred composition comprises a further growth inhibitory agent, cytotoxic agent or chemotherapeutic agent.
Another embodiment of the present invention is a method of administering a drug or a treatment comprising the steps of: a) obtaining a nucleic acid sample from an individual; b) determining the identity of the polymorphic base of at least one GSSP-2-related biallelic marker which is associated with a positive response to the treatment or the drug; or at least one biallelic GSSP-2-related biallelic marker which is associated with a negative response to the treatment or the drug; and c) administering the treatment or the drug to the individual if the nucleic acid sample contains said biallelic marker associated with a positive response to the treatment or the drug or if the nucleic acid sample lacks said biallelic marker associated with a negative response to the treatment or the drug. In addition, the methods of the present invention for administering a drug or a treatment encompass methods with any further limitation described in this disclosure, or those following, specified alone or in any combination: optionally, said GSSP-2-related biallelic marker may be in a sequence selected individually or in any combination from the group consisting of SEQ ID NOs:. 1, 2 and 4; and the complements thereof; or optionally, the administering step comprises administering the drug or the treatment to the individual if the nucleic acid sample contains said biallelic marker associated with a positive response to the treatment or the drug and the nucleic acid sample lacks said biallelic marker associated with a negative response to the treatment or the drug.
Another embodiment of the present invention is a method of selecting an individual for inclusion in a clinical trial of a treatment or drug comprising the steps of: a) obtaining a nucleic acid sample from an individual; b) determining the identity of the polymorphic base of at least one GSSP-2-related biallelic marker which is associated with a positive response to the treatment or the drug, or at least one GSSP-2-related biallelic marker which is associated with a negative response to the treatment or the drug in the nucleic acid sample, and c) including the individual in the clinical trial if the nucleic acid sample contains said GSSP-2-related biallelic marker associated with a positive response to the treatment or the drug or if the nucleic acid sample lacks said biallelic marker associated with a negative response to the treatment or the drug. In addition, the methods of the present invention for selecting an individual for inclusion in a clinical trial of a treatment or drug encompass methods with any further limitation described in this disclosure, or those following, specified alone or in any combination: Optionally, said GSSP-2-related biallelic marker may be in a sequence selected individually or in any combination from the group consisting of SEQ ID NOs:. 1, 2 and 4; and the complements thereof; optionally, the including step comprises administering the drug or the treatment to the individual if the nucleic acid sample contains said biallelic marker associated with a positive response to the treatment or the drug and the nucleic acid sample lacks said biallelic marker associated with a negative response to the treatment or the drug.
Another embodiment of the present invention is a method of determining whether an individual is at risk of developing a neoplastic disease (e.g., cancer); and determining whether the nucleotides present at one or more of the GSSP-2-related biallelic markers of the invention are indicative of a risk of developing a neoplastic disease. Optionally, said GSSP-2-related biallelic marker is a GSSP-2-related biallelic marker positioned in SEQ ID NOs: 1, 2 or 4; one or more GSSP-2-related biallelic marker selected from the group consisting of 20-828-311, 17-42-319, 17-41-250, 20-841-149, 20-842-115, and 20-853-415; or more preferably a GSSP-2-related biallelic marker selected from the group consisting of 17-42-319 and 17-41-250.
Another embodiment of the present invention is a method of determining whether an individual is at risk of developing a neoplastic disease comprising obtaining a nucleic acid sample from the individual and determining whether the nucleotides present at one or more of the polymorphic bases in a GSSP-2-related biallelic marker. Optionally, said GSSP-2-related biallelic is a GSSP-2-related biallelic marker positioned in SEQ ID NOs: 1, 2 or 4; one or more of the GSSP-2-related biallelic marker selected from the group consisting of 20-828-311, 17-42-319, 17-41-250, 20-841-149, 20-842-115, and 20-853-415; or more preferably a GSSP-2-related biallelic marker selected from the group consisting of 17-42-319 and 17-41-250.
Another embodiment of the present invention is a method of categorizing the risk of an individual developing a neoplastic disease comprising the step of assaying a sample taken from the individual to determine whether the individual carries an allelic variant of GSSP-2 associated with an increased risk of a neoplastic disease. In one aspect of this embodiment, the sample is a nucleic acid sample. In another aspect a nucleic acid sample is assayed by determining the frequency of the GSSP-2 transcripts present. In another aspect of this embodiment, the sample is a protein sample. In another aspect of this embodiment, the method further comprises determining whether the GSSP-2 protein in the sample binds an antibody specific for a GSSP-2 isoform associated with a neoplastic disease.
Another embodiment of the present invention is a method of categorizing the risk of an individual developing a neoplastic disease comprising the step of determining whether the identities of the polymorphic bases of one or more biallelic markers which are in linkage disequilibrium with the GSSP-2 gene are indicative of an increased risk of a neoplastic disease. Another embodiment of the invention encompasses the use of any polynucleotide for, or any polynucleotide for use in, determining the identity of an allele at a GSSP-2-related biallelic marker. In addition, the polynucleotides of the invention for use in determining the identity of an allele at a GSSP-2-related biallelic marker encompass polynucleotides with any further limitation described in this disclosure, or those following: Optionally, said GSSP-2-related biallelic marker is a GSSP-2-related biallelic marker positioned in SEQ ID NOs: 1, 2 or 4; one or more GSSP-2-related biallelic marker selected from the group consisting of 20-828-311, 17-42-319, 17-41-250, 20-841-149, 20-842-115, and 20-853-415; or more preferably a GSSP-2-related biallelic marker selected from the group consisting of 17-42-319 and 17-41-250. Optionally, said polynucleotide may comprise a sequence disclosed in the present specification. Optionally, said polynucleotide may consist of, or consist essentially of any polynucleotide described in the present specification. Optionally, said determining is performed in a hybridization assay, sequencing assay, microsequencing assay, or allele-specific amplification assay. Optionally, said polynucleotide is attached to a solid support, array, or addressable array. Optionally, said polynucleotide is labeled.
Another embodiment of the invention encompasses the use of any polynucleotide for, or any polynucleotide for use in, amplifying a segment of nucleotides comprising an GSSP-2-related biallelic marker. In addition, the polynucleotides of the invention for use in amplifying a segment of nucleotides comprising a GSSP-2-related biallelic marker encompass polynucleotides with any further limitation described in this disclosure, or those following: Optionally, said GSSP-2-related biallelic marker is a GSSP-2-related biallelic marker positioned in SEQ ID NOs: 1, 2 or 4; one or more GSSP-2-related biallelic marker selected from the group consisting of 20-828-311, 17-42-319, 17-41-250, 20-841-149, 20-842-115, and 20-853-415; or more prefer GSSP-2-related biallelic marker selected from the group consisting of 17-42-319 and 17-41-250 . Optionally, said polynucleotide may comprise a sequence disclosed in the present specification. Optionally, said polynucleotide may consist of, or consist essentially of any polynucleotide described in the present specification. Optionally, said amplifying is performed by a PCR or LCR. Optionally, said polynucleotide is attached to a solid support, array, or addressable array. Optionally, said polynucleotide is labeled.
An additional embodiment of the present invention is a GSSP-2 nucleic acid molecule for use in modulating apoptosis, a GSSP-2 polypeptide for use in modulating apoptosis and/or necrosis, the use of a GSSP-2 polypeptide for the manufacture of a medicament for the modulation of apoptosis and/or necrosis, and the use of a GSSP-2 nucleic acid molecule for the manufacture of a medicament for the modulation of apoptosis and/or necrosis.
Additional embodiments and aspects of the present invention are set forth in the Detailed Description of the Invention and the Examples.